Hog cholera virus or classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) belong to the genus Pestivirus of the Flaviviridae family. CSFV is restricted to swine, while BVDV and BDV have been isolated from several species such as cattle, swine, sheep, deer, and giraffes. Although pigs can be infected by all of these pestiviruses, only CSFV induces severe, often fatal, disease. The disease is characterized by fever and, for instance, leukopenia and can run an acute, chronic, or subclinical course. Although effective live-attenuated vaccines are available, pigs are not vaccinated against CSFV in the European Union (EU) because vaccinated and infected pigs are serologically indistinguishable. Outbreaks of CSF in the EU are controlled by eradication of all pigs from infected farms and farms in the vicinity. Because of this strategy, more than 10 million pigs had to be killed and destroyed during the 1997-1998 CSF epizootic in The Netherlands, costing more than $2 billion. It is for this reason that a great demand exists for a marker vaccine that provides protective immunity and induces an antibody response in the vaccinated pigs that can be distinguished from the antibody response caused by a natural CSFV infection.
Like other members of the family, pestiviruses are plus-stranded RNA viruses whose genome comprises one long open reading frame. Translation into a hypothetical polyprotein is accompanied by processing into mature proteins. The structural proteins include a nucleocapsid protein C and three envelope glycoproteins Erns, E1 and E2. The envelope proteins Erns and E2 are able to induce neutralizing antibodies. Glycoprotein E2 is the most immunogenic protein of pestiviruses and elicits high titers of neutralizing antibodies after infection. Vaccination of target animals with E2 has shown to give complete protection against a lethal homologous challenge. When E2 is used for vaccination, serological diagnosis of a natural pestivirus infection has to be performed with an immunogenic/antigenic protein other than E2 that is present in the infectious pestivirus. For this purpose, the Erns glycoprotein can be used as an antigen in a diagnostic test. A population that is vaccinated with the E2 glycoprotein can still be tested serologically for pestivirus infection with a diagnostic test based on the Erns antigen. A serological test based on Erns can distinguish Erns antibody-positive sera from animals infected with the virus and Erns antibody-negative sera from uninfected animals. This is called the marker vaccine approach. Of course, these marker vaccines depend on sensitive tests and, in the case of CSFV, the test also has to be very specific because pigs can be infected with the other pestiviruses BVDV and BDV. Because BVDV and BDV do not cause (severe) clinical symptoms in pigs and the animals are not vaccinated for these viruses, the diagnostic test for a CSFV marker vaccine should only detect CSFV antibodies and no other pestivirus antibodies.
Serological tests based on the complete Erns protein have been developed previously but are not always satisfactory in that they are not specific enough in that they cannot discriminate sufficiently between infections with different pestivirus species or are not sensitive enough to detect early infections with a pestivirus. 
In one embodiment, the invention provides a so-called transport peptide module, herein also called “movin.” In principle, we found that most linear peptides of 10 to 18 residues long which have >40% arginines (R) or lysines (K) are capable of functioning as such a transport peptide module to which cargo can be attached. Such a transport peptide module preferably should not, or only to a small extent, contain negatively charged amino acids such as aspartic acid (D) or glutamic acid (E). Preferred peptide modules are identified herein with full sequence, such as, for example, in Tables 4, 5, and 9 to 11, or retro-inverso variants thereof.
Variations in amino acid sequence are well tolerated, at least from the viewpoint of translocation as activity. As a rule of thumb, it can be said that related sequences have at least 30-50% homology, preferably at least 70% homology, and most preferably at least 85% homology, to those displayed in these tables, which allows identifying further relevant sequences present in nature or capable of being synthesized.
Substitutions in the amino acid sequence of a transport peptide module can be applied to increase the translocation (transport) activity. An optimized transport peptide module can, for example, be synthesized according to retro-inverso peptide chemistry, in which the sequence is reversed and D-amino acids are used instead of L-amino acids. Transport peptides derived from the herein-indicated positions of the Erns peptide, L3 loop peptides or human respiratory syncytial virus protein G (HRSV-G) peptides, and peptide mimics or peptoides derived thereof were able to bind surface glycosaminoglycans like heparin. Therefore, finding that a peptide belongs to the group of linear heparin-binding peptides or is capable of binding related glycosaminoglycans can be used as a prediction that they likely also can function as transport peptides. However, heparin binding is not a prerequisite for a peptide being a transport peptide.
To check if the presence of heparin on the surface of the cell influenced the efficiency of translocation, it was tested whether heparin-binding peptides also translocated into mutant cells which were glycosaminoglycan deficient (cell lines pgsA-745 and pgsD-677). Titrations of all heparin-binding peptides on the different cells showed that peptides translocated with the same efficiency/activity into heparin-containing cells and in the mutant cells without heparin (data not shown). Thus, heparin-binding peptides have translocation activity, and binding of the peptides to heparin obviously does not block the peptide from penetrating the plasma membrane. Likely, the peptides have a high on/off rate for heparin, and the high affinity for phospholipids directs the peptides to the membrane and ultimately into the cell. On the other hand, heparin binding, albeit being predictive, does not seem to be a prerequisite for efficient translocation of the peptides.
The invention further provides a method for translocating a compound over a membrane of a cell, an epithelial layer, mucus layer, blood-brain barrier or skin comprising providing the compound with a transport peptide module according to the invention and contacting it with a cell. Such compounds, herein also called cargo, can be large; successful translocation of compounds up to 600 kD has been demonstrated and it is expected that even larger compounds may be translocated. From the perspective of speed of translocation in relation to the usefulness of the compound, compounds of preferred molecular weight are those of 60 to 500 kD and even more preferred are those of 120 to 300 kD. Compounds can also be of a varied nature. For example, it is possible to link macromolecules such as nucleotides, polypeptides, drugs such as antiviral, antimicrobial or anti-inflammatory drugs, and the like to a module as provided herein for successful translocation of such a compound. Topical application of such a compound, e.g., as a pharmaceutical composition, is specifically provided. A module as provided herein has excellent capacity to penetrate to the upper layers of the skin. Typical applications include further use of a labile linker such as a thioester or a O(C═O) CH2NRC (═O) CH2NHCH2 (C═O) SCys linker. For use of a transport peptide module according to the invention, drugs or macromolecules are typically covalently coupled to the peptide, examples of which are cyclosporine A, acyclovir, and terbenafine coupled with a module according to the invention.
This invention also provides, among others, peptide-based diagnostics in connection with diseases caused by pestivirus infections. Antigenic peptides as provided herein and useful for diagnostics can surprisingly also be used otherwise, such as antibacterial or transport peptides. Because in one embodiment the transport peptide module is a fragment derived of the Erns protein, it can be used for diagnosis of pestivirus infections when a marker vaccine is used that is based on E2, another pestivirus surface protein. Due to its unique biochemical character, a peptide as provided herein has the ability to permeate and kill microorganisms and has the ability to translocate itself and a coupled cargo across a cell membrane and epithelium barrier.
In a preferred embodiment, the invention provides a thus far unidentified small, independently folding protein (peptide) module related to modules present at the C-terminal end of pestivirus Erns, at the L3 loop of secreted cytotoxic Rnases that preferably belong to the group of type II ribotoxins such as alpha-sarcin, restrictocin, mitogillin, toxin Asp fi, clavin or gigantin, in a heparin-binding peptide, in a DNA/RNA-binding peptide, in HRSV-G protein, and its use as a transport peptide. Previously, the region responsible for translocation of alpha-sarcin was thought to be located in a hydrophobic stretch, located away from the L3 loop (Mancheno et al., Biophys. J. 68, 2387-2395, 1995). In a preferred embodiment, the invention provides an isolated, synthetic or recombinant protein module or functional equivalent thereof comprising an amino acid sequence that is at least 85% identical to any of the sequences shown in Tables 1-4 and 9-11, e.g., to an amino acid sequence of a peptide located from about amino acid position 194 to 220 in a pestiviral Erns protein and/or that is at least 70% identical to an L3 loop sequence such as shown in Table 5.
Such transport peptide modules can be prepared synthetically with normal peptide synthesis or coupling techniques as described herein, starting from individual amino acids or by coupling or linking smaller peptides of relevant sequence to another or by cleaving off from larger peptides. When desired, nonconventional amino acids can be used, such as D-amino acids or others that normally do not occur in natural proteins. Peptides can also be prepared via recombinant DNA techniques via transcription and translation from recombinant nucleic acid encoding such a peptide or protein module, be it linked to, for example, a fusion protein or specific target molecule such as a desired binding molecule derived from an antibody or protein ligand or receptor-binding molecule, and so on. For example, we have successfully expressed a fusion protein of a transport peptide and Green Fluorescent protein in A72 cells. The Green Fluorescent protein showed the same cellular localization as the biotinylated transport peptide in the nucleoli and around the nucleus. This is in contrast to normally expressed Green Fluorescent protein, which was distributed evenly over the cell (data not shown).
In a preferred embodiment, the invention provides a transport peptide module or functional part thereof wherein at least the functional part of the peptide comprises a reversed amino acid sequence to one of a sequence given in claims 1 to 6 and wherein D-amino acids are used instead of L-amino acids. Reversing the sequence and using the D-amino acids instead enhances translocation activity, allowing improved use for, for example, transport of macromolecules or drugs through cell membrane barriers into cells.
In a preferred embodiment as explained herein, the invention provides a module which is functional as a transport peptide module, also when cargo is attached, wherein the peptide is located from about amino acid position 191 to 222, or from about 194 to 227, or from about 191 to 227, or from about amino acid position 176 to about 220, 222, or 227 in the case of the pestiviral Erns protein or residues 51-91 or 59-88 or from 62-88 or from 62-74, in the case of the L3 loop protein, or from about amino acid position 187 to 223 in a respiratory syncytial virus G-protein. Also, in HRSV type B, a similar region was detected from position 149 to 160 in protein G. These amino acid positions and their numbering are, of course, relative to known sequences as, for example, shown in the figures herein wherein alignments of various pestiviral sequences are shown, which, of course, allows, for example, for alignment with yet unknown pestiviral sequences and allows alignment with ribotoxin L3 loop sequences. As a rule of thumb, it can be said that related sequences have at least 30-50% homology, preferably at least 70% homology, most preferably at least 85% homology, which allows identifying further relevant sequences present in nature or capable of being synthesized. As examples herein, modules are described wherein the peptide comprises the amino acid sequence RQGAARVTSWLGRQLRIAGKRLEGRSK (SEQ ID NO:1); RQGTAKLTTWLGKQLGILGKKLENKSK (SEQ ID NO:2); RVGTAKLTTWLGKQLGILGKKLENKTK (SEQ ID NO:3); RQGAAKLTSWLGKQLGIMGKKLEHKSK (SEQ ID NO:4); GNGKLIKGRTPIKFGKADCDRPPKHSQNGMGK (SEQ ID NO:5); GDGKLIPGRTPIKFGKSDCDRPPKHSKDGNGK (SEQ ID NO:6); GEGKILKGRTPIKFGKSDCDRPPKHSKDGNGK (SEQ ID NO:7); GDGKILKGRTPIKWGNSDCDRPPKHSKNGDGK (SEQ ID NO:8); KRIPNKKPGKK (SEQ ID NO:9); KTIPSNKPKKK (SEQ ID NO:10); KPRSKNPPKKPK (SEQ ID NO:11); or a functional part thereof. However, variations can be introduced, for example, by increasing the positive charge of the peptide, preferably at positions that optimize the amphipathic nature of the peptide, but not necessarily. Another example is changing several or all L-amino acids to D-amino acids to reduce possible protease sensitivity. The translocation activity of the Erns peptide was further improved by substitution of the 2 lysines and the glutaminic acid by arginines. In a preferred embodiment, a retro-inverso variant of an above-identified peptide module is provided; such a retro-inverso peptide with an inversed sequence and D-amino acids replacing L-amino acids comprises even higher translocation activity.
Of course, the invention also provides a recombinant nucleic acid encoding a module according to the invention, for example, to provide for a proteinaceous substance provided with a module according to the invention, for example, provided with a targeting means.
The invention in one aspect also relates to the design of an antigenic substance, preferably peptide-based, corresponding to the protein module in the Erns protein of Pestiviruses or a L3 loop of ribotoxin II can be used as a basis for, e.g., diagnostics tests, antibacterial or transporter peptides. For example, in one embodiment, the invention provides a method for inducing an antibody comprising administering a module or a substance according to the invention to a host capable of forming antibodies. Antibodies can be induced classically by, for example, immunizing an animal with the antigenic substance, or via more modern techniques, such as phage display, whereby so-called synthetic antibodies are produced. Be it synthetic or classical (mono- or polyclonal), the invention provides an antibody specifically directed against a module according to the invention.
With the pestivirus-derived module and/or the antibody as provided herein, the invention provides a method for detecting the presence or absence of an antibody directed against a pestivirus in a sample comprising contacting the sample with a module or a substance according to the invention, the method preferably further comprising detecting the presence or absence of an antibody bound to the module or substance. Also provided is a method further comprising contacting the sample with the module or substance in the presence of a competing antibody directed against the module and detecting the presence or absence of competing antibody bound to the module or substance. Herewith, the invention provides use of a method according to the invention for differentiating at least one animal from at least another animal. The invention thus provides a test which is based on a small fragment of the Erns protein. Sequence analysis and homology modeling was used for pestivirus Erns to identify a region that can be used for the design of antigenic substances and resulted in the identification of a small independently folding protein module which, in its native state, is exposed on the protein surface of the complete Erns protein and can be used to design antigenic substances which are comparable or superior to the complete protein.
In a further embodiment, the invention not only provides a peptide that behaves as a superior antigen in the Erns peptide-ELISA but one that has additional characteristics that are very interesting and useful. Due to its unique biochemical nature, a peptide as provided herein, for example, corresponding to the Erns C-terminal domain or to a L3 loop in a ribotoxin, is able to interact with a cell membrane and destabilize the membrane.
The invention further provides a method for translocating a compound over a membrane of a cell, an epithelial layer, mucus layer, blood-brain barrier or skin comprising providing the compound with a module or substance or transport peptide module according to the invention and contacting it with a cell, and, furthermore, it provides a method for eliciting antibiotic activity to a microorganism comprising contacting the microorganism with the module or substance.
Herein, it is shown that such an Erns peptide or protein module as provided herein has antibacterial activity for, for example, gram-negative bacteria (E. Coli) and an L3 loop or Erns peptide and it has translocation activity for, for example, eukaryotic cell membranes. A biological membrane is a very efficient barrier that protects the micromilieu of cells or intracellular compartments from the outside milieu. In order to interfere directly with biological processes inside the cell, it is necessary that pharmaceuticals cross the lipid bilayer to block/bind their targets. Many promising, potential therapeutics (hydrophilic organic molecules, peptides, proteins or genes) are ineffective because the cell membrane forms an insurmountable barrier. However, several peptides have been discovered recently that can solve this problem because they are able to translocate over the lipid bilayer and are also able to transport a diverse set of cargos inside the cell.
Interactions of pore-forming peptides with model and artificial membranes have been studied extensively the last three decades. Several families of membrane destabilizing peptides with antitumor, haemolytic, antibacterial activity or a combination have been found. Many of these peptides form amphipathic helices with a hydrophobic face and a positive charged face that organize and aggregate on the membrane surface and destabilize the membrane. Their mode of action has some resemblance with the recently discovered transport peptides (Matsuzaki et al., Biochem. Biophys. Acta. 1376: 391-400, 1998; Lindgren et al., Trends Pharmacol. SCI 21: 99-103, 2000). The invention now provides a pharmaceutical composition comprising a module or substance according to the invention useful for several purposes. For example, the invention provides use of a module or a substance according to the invention for the preparation of a pharmaceutical composition capable of membrane translocation (a transport peptide), for the preparation of a pharmaceutical composition capable of eliciting antibiotic activity (an antibiotic), or for the preparation of a pharmaceutical composition capable of inducing antibodies (a vaccine) upon administration to a host.
The invention is further explained in the detailed description described herein without limiting it thereto.